この作品はRISystem AG、ダボス、スイスによってサポートされていました。

すべての手順はIACUCが承認したと制度ガイドライン（LandesamtエリーゼVerbraucherschutz、ZentralstelleAmtstierärztlicherDienst、ザールブリュッケン、ドイツ）を行いました。鎮痛および感染予防は、実験を行うべきであり、国や機関のそれぞれのガイドラインと一致する必要があります。 インプラントや手術器具の作製<ol><li>顕微計器箱からメスの刃（サイズ15）、小準備はさみ、細かい鉗子、ドレッシング鉗子、小さなはさみ、24ゲージ（G）と27 G針、非吸収性縫合糸5-0、および針ホルダを選択。 </li><li>髄内釘、連動ピン、特別な照準装置を箱から取り出し、ジリが見た、ジーリのテンプレートは、センタリングドリルビット（直径1mm）、ドリルビット（直径0.3mm）、およびハンドドリル（ 図を見て2;材料のリストを参照してください）。 注：intramedullary釘（直径0.8mm、15.7ミリメートルの長さ）は、大腿骨への逆行性注入のための医療グレードのステンレス鋼製の髄内釘のロックです。釘は、近位ねじ山（4ミリメートルの長さ）と、軸方向及び回転の安定性（ 図1）を達成するために、インターロックピン（直径0.3mm）を挿入するための2つの穴を有しています。 </li><li> 5分間消毒溶液（ 例えば、96％アルコール）に移植し、すべての手術器具を公開したり（蒸気滅菌、130℃、25分）、それらを滅菌します。消毒または滅菌後、無菌操作布の上に機器を配置します。小動物動作テーブルに直接隣接し、無菌操作布を置きます。 </li></ol> 2.動物、麻酔、および鎮痛<ol><li>研究と対処すべき質問のために必要に応じてマウスの系統、年齢、性別を選択してください。 注：この研究のために、12〜14週齢の雄CD-1マウスを用いました。ネイルimplaのため35グラム - ntationは、動物の理想体重は25です。 </li><li> 15ミリグラム/ kgのキシラジンおよび75 mgの/ kgのケタミンの腹腔内注射でマウスを麻酔。つま先のピンチによって麻酔を確認してください。麻酔中の乾燥から動物の目を保護するために目の潤滑剤を塗布します。麻酔の誘導後、体温を一定に保つために加熱ランプ下にマウスを置きます。 </li><li>手術後3日目までは手術の前に1日目から鎮痛のための飲料水（2.5ミリグラム/ 100ミリリットル）にトラマドール塩酸塩を適用します。 </li></ol> 3.外科的手順とネイル移植<ol><li>手術前に、全体の右後肢を剃ると脱毛クリームを適用します。 5分後、クリームを除去し、水で足をきれいに。消毒溶液（ 例えば 、96％アルコール）に移植し、すべての手術器具を公開したり（蒸気滅菌、130℃、25分）、それらを滅菌します。 </li><li>無菌条件下では、畝を配置小動物の操作テーブルの上に仰臥位で、SE。膝の顆に前方アプローチを可能にするために右ひざを曲げます。手術用メスの刃を使用して、右膝に5ミリメートル内側parapatellar切開を行います。 </li><li>細かいピンセットで膝蓋靱帯を持ち上げて、手術用メスの刃で慎重に靭帯を動員。その後、大腿骨の顆間ノッチを露出させるために、メスの刃で横方向に膝蓋骨をシフト。 </li><li>髄内腔に到達するまで穿孔することによって顆間ノッチを開きます。 <ol><li>ドリルビットを中心に1ミリメートルを使用して、大腿骨軸に対してオフセット45°で掘削を開始します。ゆっくりと、それは大腿骨の骨軸と平行になるまで掘削中にドリルビットの方向を変えます。髄内空洞に達した場合はドリル停止します。 </li></ol></li><li>顆間窩に骨を開いた後、大腿骨の全長にわたって髄内キャビティに24 G針を挿入します。 intrameを連手動で24 G針の回転運動を通して大腿骨のdullary空洞。 24 G針を外し、髄内キャビティにシンナー27 G針を挿入します。大転子で近位大腿骨の皮質骨を穿孔するために前方に針を押してください。 </li><li>大腿骨から27 G針を外します。爪の先端が顆のレベルに達するまで、ハンドドリルを使用して、連続的な回転及び軸方向圧下顆間ノッチを通って髄内釘を打ち込みます。 注：爪の先端が小さいマークで識別することができます。 </li><li>左側臥位にマウスを置きます。外科的に大腿骨の中間シャフトを露出させるために股関節に膝関節からの横方向の大腿骨の骨幹部分に沿ってメスの刃を使用して、縦方向の皮膚切開を行います。 </li><li>小さな準備ハサミを使用して、筋膜を分割し、側方からの大腿骨軸の方向に筋肉を広げました。大腿骨の骨幹の一部が露出するまでの筋肉を広げます。坐骨神経を保持します。 <ol><li>ドレッシング鉗子で骨を損なうことにより、大腿骨の全周を準備します。次に、ドレッシング鉗子を広げることによって筋肉を撤回し、大腿骨を露出させます。 </li></ol></li><li>爪の先端に照準装置をマウントします。それは爪のアダプタフランジに取り付けられるまで、デバイスを前進させ、大腿骨の前外側の位置に照準装置の電源を入れます。 </li><li>近位および遠位連動ピンと釘を連動。 <ol><li>近位の連動ピンと起動します。 </li><li>ハンドドリルにセンタリングドリルビット（直径1mm）を挿入します。近位の連動穴位置で骨を皿穴。 注：皿穴により、小さな空洞が骨ドリルスルーせずに面した皮質骨に作成されます。この空洞は、薄いドリルビット（直径0.3mm）の改善されたセンタリングと案内が可能になり、後で使用。 </li><li>ハンドドリルにドリルビット（直径0.3mm）を挿入します。照準装置を使用して、対面および回避皮質骨（bicortical）の両方に穴を開けます。照準装置を介して第1連動ピンを挿入します。すぐに連動トルクが達成されるようにオフ連動ピン駆動軸ばさみ。 </li><li>遠位連動ピンに対して、この手順を繰り返します。 </li></ol></li><li>骨幹骨切り術を行います。 <ol><li> 2インターロックピン間の側面に照準装置に鋸ガイドを取り付けます。そして、ジリと骨は生理食塩水で連続灌漑の下で見た見ました。骨切り術が完了した後、骨に近い、一端でのこぎりをカット。軟組織への損傷を引き起こすことを避けるために慎重にのこぎりを削除します。 </li></ol></li><li>標線で髄内釘の残りのシャフトをオフクリップ、小さなペンチで、照準装置を取り外します。 </li><li>緯度で筋肉層を閉じます大腿骨のERALサイトと単一縫合糸で皮膚閉鎖を行います。膝の前方サイトでは、膝蓋骨の位置を変更し、単一の縫合糸で筋肉に膝蓋骨腱を固定します。同様に、この傷を閉じるために、単一の縫合糸を使用してください。 </li><li>彼らは麻酔から回復するまで加熱ランプの下で動物を保管してください。彼らは腹側横臥位を維持するのに十分な意識を取り戻してきたまでは無人の動物を放置しないでください。動物施設内の単一のケージに動物を返します。 </li><li>毎日慎重に動物を監視します。最初の3日間に術後鎮痛を維持します。発声、落ち着きのなさ、移動性の欠如、グルーミングの失敗、異常な姿勢、そして周囲の正常な関心の欠如によって示されるようにあれば鎮痛を続行し、手術後4日目に、動物はまだ、痛みの証拠を示します。動物が痛み自由であるとき鎮痛を終了します。 </li></ol>

<ol>
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Romano Matthys is an employee of RISystem AG, Davos, Switzerland, which produces the implants and implant-specific instruments. The other authors have no conflicts of interest.

外科技術の最も重要なステップは、爪の正確な位置決め、照準装置、およびピンです。爪は、顆のレベルで膝関節への爪の突起が膝（ 図3 A）の動きを制限することができるので、爪の先端にマークされたインデントに完全に挿入しなければなりません。したがって、大腿骨の大きさとは、従って、動物の体重を考慮しなければなりません。外科医はまた、爪のアダプタフランジ?...

骨治癒の生物学、細胞およびスフェロイド培養物を用いてインビトロで研究することができるが、それはまた、動物研究を使用して近づく、インビボで必要です。大動物実験はまだ前臨床試験において重要な役割を果たしているが、製品や仮説の初期段階のテストでは、過去10年の間に変更されており、今日では多くの場合、小動物モデル1で行われます。このスイッチは、いくつかの理由のために実施しました。マウスおよびラットの生産とメンテナンスは豚や羊に比べて安くなっています。また、小動物は、慢性実験の大規模シリーズの性能を容易にどちらも短い再生時間と短く、通常の治癒期間を有します。最後に、遺伝子標的動物と特異的抗体の利用可能性は、骨治癒の分子メカニズムの分析を可能にします。以前より大きな動物モデルにおいて骨接合技術を使用しながら、しかし、最小限のvariatで翻訳することができました<html>ヒトまたは動物の臨床患者のケアに使用される同様の手順からのイオンは、小型ラットおよびマウスにおける骨接合技術の開発及び適用は、困難であることが判明しました。 十分に生体力学的環境が著しく骨治癒プロセス2に影響を与えることが知られています。 micromovementsと剛性の低い固定後の剛性固定および軟骨内骨化後の膜内骨化を含む、ヒトにおける骨折治癒、治癒の異なるモードでの骨折安定化結果の違いから知られているように。完全な軸または回転不安定性は、治癒プロセスを遅延させることができるか、非組合3になることがあります。従って、我々は、マウスおよびラットで洗練されたインプラントシステムと骨接合技術の開発が必要であると感じています。このようにして、生体力学的条件は、治癒過程を分析するときに有効な結果を保証し、適切に標準化することができます。 e_content &quot;&gt;高度に洗練されたネズミの安定化技術の相当数は、ここ数年の間に導入されているが、最も一般的に使用される技術はまだ簡単な髄内ピンである。この技術の主な欠点は、しかし、回転および軸方向の欠如であります安定性4。回転および軸方向の安定性を向上させるために、髄内スクリューは、マウス5に大腿骨の骨折を安定化するために導入された。しかし、ねじ固定が原因で骨片の間の接触および圧縮の必要性骨欠損の治癒を分析するために使用することができません回転安定性を維持するために。 爪をロック髄内はシンプルなピンと髄内スクリュー4と比較して、より高い軸方向および回転安定性を提供しています。なぜならジーリのためのガイドの可能性の高い再現性大腿骨骨切り術は、見て、定義されたギャップサイズを作成する機能は、通常のボン両方の分析を可能にします電子治癒および骨欠損の治癒6。インターロックピンを挿入する、爪をロック髄内は、全重量を保有しながらも、全体の治癒過程の間に一定のギャップの大きさを保証します。ここでは、正常および遅延骨治癒に関する実験的研究で、その長所と短所の髄内固定釘の設計と応用について報告するだけでなく、。

<table border="0" cellpadding="0" cellspacing="0" width="476">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:187px;">MouseNail</td>
			<td style="width:179px;">RISystem AG</td>
			<td style="width:111px;">221,122</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MouseNail aiming device</td>
			<td>RISystem AG</td>
			<td>221,201</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MouseNail interlocking pin</td>
			<td>RISystem AG</td>
			<td>221,121</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Centering bit</td>
			<td>RISystem AG</td>
			<td>592,205</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Drill bit</td>
			<td>RISystem AG</td>
			<td>590,200</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Gigli wire saw</td>
			<td>RISystem AG</td>
			<td>590,100</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Suture (5-0 Prolene)</td>
			<td>Ethicon</td>
			<td>8614H</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Forceps</td>
			<td>Braun Aesculap AG &#38;CoKG&#160;</td>
			<td>BD520R</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dressing forceps</td>
			<td>Braun Aesculap AG &#38;CoKG&#160;</td>
			<td>BJ009R</td>
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			<td height="17" style="height:17px;">Scissors</td>
			<td>Braun Aesculap AG &#38;CoKG&#160;</td>
			<td>BC100R</td>
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			<td height="17" style="height:17px;">Needle holder</td>
			<td>Braun Aesculap AG &#38;CoKG&#160;</td>
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			<td>BD Mircolance 3</td>
			<td>304100</td>
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			<td>Braun Melsungen AG</td>
			<td>9186182</td>
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			<td>Braun Aesculap AG &#38;CoKG&#160;</td>
			<td>16600525</td>
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			<td>Knipex</td>
			<td>7932125</td>
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			<td height="17" style="height:17px;">Heat radiator</td>
			<td>Sanitas</td>
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			<td height="17" style="height:17px;">Depilatory cream</td>
			<td>Asid bonz GmbH</td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; NDXZ10</td>
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			<td height="17" style="height:17px;">Eye lubricant</td>
			<td>Bayer Vital GmbH</td>
			<td>2182442</td>
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			<td height="17" style="height:17px;">Xylazine</td>
			<td>Bayer Vital GmbH</td>
			<td align="right">1320422</td>
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			<td height="17" style="height:17px;">Ketamine</td>
			<td>Serumwerke Bernburg</td>
			<td align="right">7005294</td>
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			<td height="17" style="height:17px;">Tramadol</td>
			<td>Gr&#252;nenthal GmbH</td>
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			<td height="17" style="height:17px;">Disinfection solution (SoftaseptN)</td>
			<td>Braun Melsungen AG</td>
			<td align="right">8505018</td>
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			<td height="17" style="height:17px;">CD-1 mice</td>
			<td>Charles River</td>
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an intramedullary locking nail for standardized fixation of femur osteotomies to analyze normal and defective bone healing in mice

骨治癒モデルは、臨床的骨折の治療のための新しい治療戦略の開発に不可欠です。さらに、マウスモデルは、外傷の研​​究に使用されるより一般的になってきています。彼らは、骨治癒の高度に差別化プロセスの背後にある分子機構の解析のための変異株と多数の抗体を提供しています。生体力学的環境を制御するために、標準化された、よく特徴付けられた骨接合技術は、マウスでは必須です。ここでは、マウスにオープン大腿骨骨切り術を安定化させるための髄内釘の設計および使用について報告します。医療グレードのステンレス鋼製の釘は、高い軸方向と回転剛性を提供します。さらにインプラントは定義され、一定の骨切り術ギャップの作成は0.00ミリメートルから2.00ミリメートルにサイズができます。 0.00ミリメートルと0.25ミリメートルのギャップサイズの大腿骨骨切り術の爪の安定化をロックする髄内には、軟骨内および膜内ossificatを通じて十分な骨の治癒をもたらしますイオン。萎縮性非組合で2.00ミリメートル結果のギャップサイズと大腿骨骨切り術の安定化。従って、髄内釘のロックは、治癒および非治癒モデルで使用することができます。他のオープン骨治癒のモデルと比較して、爪の使用のさらなる利点は、十分に骨統合のプロセスを研究するために、代用骨と足場を固定するための可能性です。髄内釘の使用の欠点は、閉じられたモデルと比較すると、開いているすべての手順に固有のより侵襲的な外科的手技、です。さらなる欠点は、髄外安定化手順と比較して、すべての髄安定化技術に固有の髄内腔に何らかの損傷を誘導することができます。

外科的処置のための全体的な時間は、創傷閉鎖するために皮膚切開から約30分でした。提供される外科用インプラントを使用して、外科手術は、ステレオ顕微鏡なしで行うことができます。手術後、動物を毎日モニターしました。動物のどれもが、この期間の後の痛み（発声、落ち着きのなさ、移動性の欠如、グルーミングの失敗、異常な姿勢、または周囲の正常な?...

Watch this Scientific Journal Video about An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice at JoVE.com

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

マウスにおける正常および不良骨治癒を分析するために大腿骨骨切り術の標準化された固定用髄内ロッキングネイル

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming ...

an-intramedullary-locking-nail-for-standardized-fixation-femur

Research

JoVE Journal

Medicine

13.3K ビュー.  Saarland University. This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

ビデオ: An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

競争力のあるマウス骨髄移植モデルにおけるドナー細胞の生着を​​決定するために、定量的リアルタイムPCRを用いて

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

医学

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are exacerbated when significant brain atrophy and age-related white matter changes (e.g. Leukoaraiosis) are present. Lesion Explorer (LE) is an accurate and reliable neuroimaging pipeline specifically developed to address such issues commonly observed on MRI of Alzheimer's disease and normal elderly. The pipeline is a complex set of semi-automatic procedures which has been previously validated in a series of internal and external reliability tests1,2. However, LE's accuracy and reliability is highly dependent on properly trained manual operators to execute commands, identify distinct anatomical landmarks, and manually edit/verify various computer-generated segmentation outputs.
LE can be divided into 3 main components, each requiring a set of commands and manual operations: 1) Brain-Sizer, 2) SABRE, and 3) Lesion-Seg. Brain-Sizer's manual operations involve editing of the automatic skull-stripped total intracranial vault (TIV) extraction mask, designation of ventricular cerebrospinal fluid (vCSF), and removal of subtentorial structures. The SABRE component requires checking of image alignment along the anterior and posterior commissure (ACPC) plane, and identification of several anatomical landmarks required for regional parcellation. Finally, the Lesion-Seg component involves manual checking of the automatic lesion segmentation of subcortical hyperintensities (SH) for false positive errors.
While on-site training of the LE pipeline is preferable, readily available visual teaching tools with interactive training images are a viable alternative. Developed to ensure a high degree of accuracy and reliability, the following is a step-by-step, video-guided, standardized protocol for LE's manual procedures.

Lesion Explorer (LE) is a semi-automatic, image-processing pipeline developed to obtain regional brain tissue and subcortical hyperintensity lesion volumetrics from structural MRI of Alzheimer's disease and normal elderly. To ensure a high level of accuracy and reliability, the following is a video-guided, standardized protocol for LE's manual procedures.

病変エクスプローラ：アルツハイマー病における正確で信頼性のMRI誘導Volumetrics用ビデオガイド下、標準化されたプロトコルおよび標準高齢者

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are ...

Adjustable Stiffness, External Fixator for the Rat Femur Osteotomy and Segmental Bone Defect Models

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there has been great clinical interest in improving bone healing by altering the mechanical environment through the fixation stability around the lesion. One constraint of preclinical animal research in this area is the lack of experimental control over the local mechanical environment within a large segmental defect as well as osteotomies as they heal. In this paper we report on the design and use of an external fixator to study the healing of large segmental bone defects or osteotomies. This device not only allows for controlled axial stiffness on the bone lesion as it heals, but it also enables the change of stiffness during the healing process in vivo. The conducted experiments have shown that the fixators were able to maintain a 5 mm femoral defect gap in rats in vivo during unrestricted cage activity for at least 8 weeks. Likewise, we observed no distortion or infections, including pin infections during the entire healing period. These results demonstrate that our newly developed external fixator was able to achieve reproducible and standardized stabilization, and the alteration of the mechanical environment of in vivo rat large bone defects and various size osteotomies. This confirms that the external fixation device is well suited for preclinical research investigations using a rat model in the field of bone regeneration and repair.

One constraint of preclinical research in the field of bone repair is the lack of experimental control over the local mechanical environment within a healing bone lesion. We report the design and use of an external fixator for bone repair with the ability to change fixator stiffness in vivo.

調整可能な剛性、ラット大腿骨骨切り術や分節骨欠損モデルの創外固定器

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

合わせた<em&gt;インビボ</em&gt;光とμCTイメージングマウスにおける整形外科インプラント感染で感染症、炎症、および骨解剖学を監視する

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

生きたマウスにおける大腿骨髄吸引

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

マウスの気管の迅速な挿管のための改善された方法

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

正常マウスで70％肝切除後の血管と実質再生の可視化

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

生体内で破壊カルス開発マウス大腿骨の骨、MRI 対応のデバイスを用いたマウスの骨治癒過程の評価

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

癒しの標準化された生体力学的条件下でマウスの軟骨破壊を分析する低侵襲モデル

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

角膜上皮摩耗眼はりのモデルとして角膜創傷治癒

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...